Clostridium difficile is the major cause of nosocomial, antibiotic-associated diarrhea, an emerging drug- resistant infection. The antibiotic armamentarium for treating C. difficile infection (CDI) is limited and inadequate: although vancomycin and metronidazole are effective for initial disease, they are associated with high rates of relapse or re-infection. As a result, new drugs for C. difficile are desperately needed. Sequella, Inc. discovered a new class of small molecule ethylenediamine compounds with a novel mechanism of antibacterial activity. SQ109, one of the compounds of this class, is in clinical trials for treatment of tuberculosis (TB). Phase 1a and b studies demonstrated safety in humans, and Phase 2 efficacy trials will begin in fall 2010. In addition to anti-TB activity, SQ109 has in vitro activity against C. difficile, and we propose to use this lead compound as a foundation to the optimization of an ethylenediamine(s) for treatment of CDI. In Aim 1, we will (a) synthesize 220-240 ethylenediamine compounds based on SQ109 structure and (b) evaluate them in a series of assays for in vitro activity against C. difficile (determination of the Minimum Inhibitory Concentration, MIC), mammalian cell cytotoxicity, and ability to block C. difficile toxin release. Based on structure-activity relationships, we will synthesize and evaluate additional compounds. In Aim 2, the most promising 15-20 compounds identified in Aim 1 will be evaluated in additional studies, including determination of the maximum tolerated dose in mice, in vivo efficacy in animal models of infection, activity against intestinal flora (to evaluate specificity). The most promising compounds will also undergo predictive Absorption, Distribution, Metabolism, Excretion, and Toxicity studies. Again, we may synthesize and evaluate additional compounds based on SAR data obtained in this aim. In Aim 3, a more in-depth series of studies will be implemented for the 5 best compounds, which include additional in vivo efficacy studies, in vivo pharmacokinetic (PK) evaluations, spectrum of activity, synergy with other drugs, and determination of the frequency of resistance development in vitro. Finally, in Aim 4, we will select the best compound on which to perform additional in vivo studies, evaluate drug candidate effects on the microbiome, and perform PK/pharmacodynamic testing and dose-finding toxicity studies. At the conclusion of this grant, our goal is to have identified the ethylenediamine that is best-in-class for the treatment of CDI. We will have performed all studies necessary to commence IND-directed preclinical studies, with the ultimate goal of filing an IND with the FDA for the clinical testing of a new drug candidate for CDI.